Types Of Amino Acids Research Articles

Progress toward automated methyl assignments for methyl-TROSY applications

Methyl-TROSY spectroscopy has extended the reach of solution-state NMR to supra-molecular machineries over 100kDa in size. Methyl groups are ideal probes for studying structure, dynamics, and protein-protein interactions in quasi-physiological conditions with atomic resolution. Successful implementation of the methodology requires accurate methyl chemical shift assignment, and the task still poses a significant challenge in the field. In this work, we outline the current state of technology for methyl labeling, data collection, data analysis, and nuclear Overhauser effect (NOE)-based automated methyl assignment approaches. We present MAGIC-Act and MAGIC-View, two Python extensions developed as part of the popular NMRFAM-Sparky package, and MAGIC-Net a standalone structure-based network analysis program. MAGIC-Act conducts statistically driven amino acid typing, Leu/Val pairing guided by 3D HMBC-HMQC, and NOESY cross-peak symmetry checking. MAGIC-Net provides model-based NOE statistics to aid in selection of a methyl labeling scheme. The programs provide a versatile, semi-automated framework for rapid methyl assignment.

Comparison the visible photocatalytic activity and kinetic performance of amino acids (non-metal doped) TiO2 for degradation of colored wastewater effluent

Different types of amino acids as sources of carbon, nitrogen and sulfur were doped on TiO2 lattice to study the photocatalytic activity of three synthesized photocatalysts for dye removal under visible light. The phase composition, functional groups, morphology, optical properties, band gap energy and recombination rate of nanostructures were studied by X-ray diffraction (XRD), fourier transform infrared (FT-IR), field emission scanning electron microscopy with Energy Dispersive X-Ray Spectroscopy (FESEM/EDX), diffuse reflectance spectra (DRS) and photoluminescence spectroscopy (PL). These analysis verified the successful synthesis of the catalysts and showed the type of catalyst significantly affected PL analysis as l-Arginine-TiO2 (C,N co-doped TiO2) had lower recombination rate and PL intensity compared to l-Proline-TiO2 (C,N co-doped TiO2) and l-Methionine-TiO2 (C,N,S tri-doped TiO2). The firs-order kinetic model described the photodegradation of methyl orange (MO) and direct red 16 (DR16) and the rate constant for DR16 photocatalytic removal using l-Arginine (1 wt %)-TiO2 was 2.9 and 4.3 times higher compared to those of l-Methionine (1.5 wt %.)-TiO2 and l-Proline (2 wt %.)-TiO2 respectively. The effect of five processing variables and their optimum values were determined using central composite design (CCD). The result confirmed that decreasing pollutant concentration and pH and increasing catalyst concentration, irradiation time and light intensity improved the removal efficiency. The catalyst showed reusable and stable structure as 93% and 84% of DR16 and MO removed after four times of reusing.

Preparation and antibacterial properties of hemp cellulose-based material based on Schiff base between lysine grafted N-halamine and dialdehyde hemp

Bacteria have exerted a great threat to the human living environment. Therefore, many industries put forward higher requirements for the antibacterial properties of cellulose-based materials, especially the applications in food packaging, biomedical, and textile industry. In this paper, through using synthetic lysine grafted N-halamine organic antibacterial agent and hemp fiber as raw materials, the lysine grafted N-halamine Schiff base type hemp cellulose-based antibacterial material is prepared by simple and mild Schiff base reaction. This hemp cellulose-based antibacterial material combines various sterilization methods of hemp fiber, amino acid type Schiff base and N-halamine. The synergistic cooperation of different sterilization methods greatly improves the antibacterial performance of material and reduces the possible bacterial resistance. The antibacterial examination shows that S. aureus and E. coli could be killed totally by this material in 5 min, and the inhibition zone is increased to 18.4 mm. Meanwhile, this hemp cellulose-based antibacterial material has low cost, green preparation, excellent storage stability and regeneration ability, which provides a novel way for the development of synergistic application of different antibacterial agents and the use of cellulose-based materials in bacterial environment.

Arginine: What You Need to Know for Pressure Injury Healing.

To provide information about arginine, its metabolism, and its role in acute and chronic wound healing, to assist providers in understanding the recommendations for arginine supplementation. This continuing education activity is intended for physicians, physician assistants, nurse practitioners, and nurses with an interest in skin and wound care. After participating in this educational activity, the participant will:1. Describe the characteristics of arginine.2. Choose the metabolic processes that define arginine's role in wound healing.3. Identify the average daily intake of arginine in an American diet.4. Select the evidence that demonstrates the effectiveness of arginine supplementation for wound healing. Nutrition has an important and integral role in wound healing. Arginine, a type of indispensable amino acid, has long been thought to have wound healing properties. The 2019 international guideline by the European Pressure Ulcer Advisory Panel, National Pressure Injury Advisory Panel, and Pan Pacific Pressure Injury Alliance recommends use of a high-protein, high-calorie oral nutrition supplement fortified with arginine and other antioxidants to treat adults with stage 2 or greater pressure injury and who are malnourished or at risk of malnutrition to foster healing. This article provides necessary background on this conditionally indispensable amino acid, its metabolism, and its role in acute and chronic wound healing to assist providers in understanding the recommendation for arginine supplementation.

Selective 1 Hα NMR Methods Reveal Functionally Relevant Proline cis/trans Isomers in Intrinsically Disordered Proteins: Characterization of Minor Forms, Effects of Phosphorylation, and Occurrence in Proteome.

It is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins, and to characterize minor species that are present due to the conformational heterogeneity in intrinsically disordered proteins (IDPs). To obtain residue level information on these mobile systems we introduce two 1Hα‐detected, proline selective, real‐time homodecoupled NMR experiments and analyze the proline abundant transactivation domain of p53. The measurements are sensitive enough to identify minor conformers present in 4–15 % amounts; moreover, we show the consequences of CK2 phosphorylation on the cis/trans‐proline equilibrium. Using our results and available literature data we perform a statistical analysis on how the amino acid type effects the cis/trans‐proline distribution. The methods are applicable under physiological conditions, they can contribute to find key proline isomers in proteins, and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes.

Selective 1Hα NMR Methods Reveal Functionally Relevant Proline cis/trans Isomers in Intrinsically Disordered Proteins: Characterization of Minor Forms, Effects of Phosphorylation, and Occurrence in Proteome

AbstractIt is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins, and to characterize minor species that are present due to the conformational heterogeneity in intrinsically disordered proteins (IDPs). To obtain residue level information on these mobile systems we introduce two 1Hα‐detected, proline selective, real‐time homodecoupled NMR experiments and analyze the proline abundant transactivation domain of p53. The measurements are sensitive enough to identify minor conformers present in 4–15 % amounts; moreover, we show the consequences of CK2 phosphorylation on the cis/trans‐proline equilibrium. Using our results and available literature data we perform a statistical analysis on how the amino acid type effects the cis/trans‐proline distribution. The methods are applicable under physiological conditions, they can contribute to find key proline isomers in proteins, and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes.

Solid-state fermentation by Aspergillus niger and Trichoderma koningii improves the quality of tea dregs for use as feed additives.

This study evaluated the ability of Aspergillus niger and Trichoderma koningii to improve the quality of tea dregs (TDs) through solid-state fermentation as well as the value of the fermented tea dregs (FTDs) produced for use as bio-feed additives. After fermentation, FTDs differed in color and structure. Fermentation with A. niger and T. koningii increased the contents of crude protein, crude fiber, neutral detergent fiber, and acid detergent fiber of TDs. Compared to the unfermented group, the contents of reducing sugar, total flavonoids, total polyphenols, and theasaponins were increased in A. niger FTDs, while in T. koningii FTDs caffeine was completely degraded, the theasaponins were lower, and the contents of reducing sugar and caffeine higher. Regarding free amino acids, A. niger FTDs had the highest content of total amino acids, total essential amino acids, total non-essential amino acids, total aromatic amino acids, total branched-chain amino acids, and total non-protein amino acids, and all types of essential amino acids, followed by T. koningii FTDs and the control TDs. Fungal fermentation had similar effects on the content of various hydrolytic amino acids as those on above free amino acids, and increased the content of bitter and umami components. The composition of essential amino acids of TDs or FTDs was similar to that of the standard model, except for sulfur-containing amino acids and isoleucine. Solid-state fermentation with A. niger and T. koningii effectively improved the nutritional value of TDs, increased the contents of functional substances, and improved the flavor of TDs. This study demonstrated a feasible approach to utilize TDs that not only increases animal feed resources, but also reduces the production of resource waste and pollution.

An Insight into the Proofreading Functions of Multisubunit DNA-Dependent RNA Polymerases and Their Catalytic Mechanism

Aim: To analyze the active sites of the proofreading (PR) functions in the multisubunit DNA-dependent RNA polymerases (MSU RNAPs) from prokaryotes, chloroplasts and eukaryotes, and propose a plausible unified catalytic mechanism for these enzymes.
 Study Design: Data collected on these enzymes from bioinformatics, biochemical, site-directed mutagenesis (SDM), X-ray crystallography and cryo-electron microscopy (cryo-EM) were used for the analyses.
 Methodology: The protein sequence data of MSU RNAPs from prokaryotes, prokaryotic-types (plant chloroplasts) and eukaryotes were obtained from PUBMED and SWISS-PROT databases. The advanced version of Clustal Omega was used for protein sequence analysis. Along with the conserved motifs identified by the bioinformatics analysis, the data already available from biochemical and SDM experiments, and X-ray crystallographic and cryo-EM data on these enzymes are also used to confirm the possible amino acids involved in the active site of the PR function in these MSU RNAPs
 Results: All the seven types of MSU RNAPs (I-VII) reported from prokaryotes to eukaryotes were analyzed by the multiple sequence alignment (MSA) software, Clustal Omega, to find out conservations among them. The MSA analysis showed many conserved amino acid motifs including small and large peptide regions from the MSU RNAPs of prokaryotes, eukaryotes and plant chloroplasts. Interestingly, the catalytic amino acid and template-binding pairs are highly conserved in all these polymerases, with a few exceptions. Most of them use a basic amino acid (R/K/H) for initiating catalysis and an -YG/FG- pair for template-binding. Some odd type of catalytic amino acids and template-binding pairs are observed in human pathogens, parasites and organisms which cannot ferment sugars. In all the MSU RNAPs, the proposed polymerase catalytic region also possessed three invariant Cs and an invariant H within it. The invariant Cs is shown to bind a zinc atom and proposed to involve in the PR function by excising any misincorporated nucleotide during the transcription process. In the plant-specific MSU RNAPs IV and V, which involve in transcriptional gene silencing in plants, the catalytic and template-binding pairs do not follow the regular distance conservations as observed with other five of the MSU RNAPs. Their polymerase/PR active site regions are similar to RNAP III rather than to RNAP II, as all three make only low molecular weight RNAs.
 Conclusions: All the known MSU RNAPs possess three invariant Cs and an invariant H embedded within the polymerase active site itself. The three invariant Cs are shown to bind a zinc atom and the invariant H could act as the proton acceptor from a metal-bound water molecule, for initiating excision of the mismatches by a Zn-mediated hydrolysis. Thus, the PR function in MSU RNAPs is integrated within the polymerase active site itself, which is in sharp contrast to the PR functions reported in DNA-dependent DNA polymerases and RNA-dependent RNA polymerases. Therefore, all the seven MSU RNAPs from prokaryotes and eukaryotes are proposed to follow a unified mechanism to excise the mismatches during transcription. The discovery of intrinsic self-correcting RNA transcription mechanism fulfils the missing link in molecular evolution.

Synthesis and bioactivities of new N-terminal dipeptide mimetics with aromatic amide moiety: Broad-spectrum antibacterial activity and high antineoplastic activity

The increasingly growing epidemics of multidrug-resistant bacteria are becoming severe public health threat. There is in an urgent need to develop new antibacterial agents with broad-spectrum antibacterial activity and high selectivity. Here, a series of N-terminal dipeptide mimetics with an aromatic amide moiety were synthesized from amino acids. The effects of amino acid type and aromatic moiety on the biological activities of the mimetics were evaluated. The dipeptide mimetics not only showed significant broad-spectrum antibacterial activity against Gram-negative (Escherichia coli and Klebsiella pneumoniae), Gram-positive (Staphylococcus aureus) and drug-resistant bacterium MRSA (methicillin-resistant S. aureus) but also demonstrated high selectivity for S. aureus versus mammalian erythrocytes. The coupling product of L-valine with p-alkynylaniline (dipeptide mimetic 7) exhibited the best antibacterial activities with minimum inhibitory concentration (MIC) ranging from 2.5 to 5 μg/mL. Moreover, the bactericidal kinetics and multi-passage resistance tests indicated that the mimetic 7 both rapidly killed bacteria and had a low probability of emergence of antimalarial resistance. Meanwhile, the mimetic 7 possessed the ability to both inhibit bacterial biofilm formation and eradicate mature biofilm. The depolarization and destruction of the bacterial cell membrane is the main sterilization mechanism, which hinders the propensity to develop bacterial resistance. Furthermore, the mimetic 7 also showed good antineoplastic activity against gastric cancer cell (SGC 7901, IC50 = 70.8 μg/mL), while it had very low toxicity to mammalian cell (L929). The mimetics bear considerable potential to be used as antibacterial and anticancer agents to combat antibiotic resistance.

The Profile of Amino Acids in Sea Horse ((Hippocampus sp)

Seahorse (Hippocampus sp.) is a marine fisheries commodity that has high economic and nutritional values. The aims of study were to determine the proximate composition and the profile of the amino acids content in seahorses. The method used refers to the AOAC (Association of Official Analytical Chemistry) for proximate analysis and a GC-MS (Gas Chromatography-Mass Spectroscopy) for the amino acid analysis. This research was done in March 2021. The sea horses were carried out from Simeulue waters, Simeulue district, Aceh Province, Indonesia. Samples were divided into 2 sizes, small size (3.00-5.00 cm) and large size ((5.01-10.00 cm). The analysis was done at laboratorium of Syiah Kuala University. The results of the study was found that the proximate content of the seahorse was water content (6.29%), ash content (25.43%), fat content (0.57%), protein content (39.32%) and carbohydrates ( 28.48%). The amino acid composition of large seahorses and small seahorses consists of four types of essential amino acids, namely leucine, isoleucine, histidine and valine, and six types of non-essential amino acids, namely alanine, glycine, aspartic acid, asparagine, arginine and serine.

Manufacture of Toman Fish Protein (Channa micropeltes) Isolate with Different pH Methods using Star Fruit (Averrhoa bilimbi) Acid

Toman fish (Channa micropeltes) is a type of freshwater fish that belongs to the genus Channa which is the largest size compared to the others and has high protein. This study aims to obtain the isolate of toman fish protein (Channa micropeltes) by the method of pH regulation using star fruit acid. Wuluh star fruit acid is used as a substitute for HCl in making fish protein isolates to reduce pH. This research method uses a Completely Randomized Design (CRD). The treatments consisted of (P1 = pH 4, P2 = pH 5, P3 = pH 6). The results showed that star fruit acid can replace HCl as a protein precipitant and P1 (pH 4) obtained the most isolates, namely an average protein content of 91.64% (db), a moisture content of 1.67%, ash content of 2.06 % (db), fat content of 2.74% (db), yield of 88.46%, and total amino acid composition of 61.30%, where the type of amino acid that dominates is glutamic acid (12.30%). Glutamic acid amino acid is the one that influences a lot in giving pH isoelectric point, in the treatment of P1 with pH 4 making isolates of toman fish protein to be the best treatment, because glutamic acid has an isoelectric point of 3.22 which is the closest to pH 4 plus also the pH of isoelectric point from other amino acids.

Achieving a highly efficient chitosan-based triboelectric nanogenerator via adding organic proteins: Influence of morphology and molecular structure

The utilization of biodegradable materials for the triboelectric nanogenerator (TENG) has provoked intensive interest since the growth of electronic devices tended to operate in proximity of the human body. This work proposed highly efficient biopolymer, based on a chitosan (CS) TENG, by incorporating protein-based compounds as fillers. The effect of different types of protein; albumin, egg-shell membrane (ESM) and silk fiber (SF), with the loaded content on the output performance of TENG was explored. It was found that the output signal can be maintained and even enhanced by embedding interlaced-fiber and making a gauze-like structure inside the CS matrix. Morphologies not only have different types of amino acid side chains, but also affect triboelectric output performance. High content of glycine, alanine and serine consequently increases the output signal by providing additional charges from molecular polarizations. Mixing glycine amino acid in the presence of alanine and serine reduces the centrosymmetric structure of the host amino acid and creates polar domains. Adding SF in CS, therefore, achieves the best output voltage ( V OC ) and current ( I SC ) as compared to other additives. The CS/SF TENG, with 10 wt% SF added, can reach ~ 77 V and ~ 13 µA of V OC and I SC , respectively. Its maximum output power of ~ 202 μW, with power density of 22.4 μW/cm 2 , is 6 times higher than that from pristine CS TENG, which can easily light up 59 light emitting diodes (LEDs) connected in series. Finally, the biodegradability was confirmed at various weeks related to the output measurement of V OC and I SC . After 9 weeks of the soil burial test, the CS/SF still generated ~ 20 V V OC and ~ 2.5 µA I SC with 30% weight loss. • The highly efficient chitosan-based TENG is fabricate by adding organic proteins as fillers. • Molecular polarizations in organic proteins boost the powder density. • The amino acid types and their concentration were proven to have the crucial role in triboelectric performance. • The biodegradability was confirmed at various weeks related to the output measurement of V OC and I SC .

Response of Cherry Tomato to Foliar Spray of Amino Acid, Bio-Catalyst and Dotted Type in Qualities and Evaluation of Drip Irrigation System

The experiment was conducted in the winter season of 2020 at the Agricultural Research and Experimentation Station of the Faculty of Agriculture, the University of Kirkuk under non-warm plastic house conditions and the study included three factors, the first factor, the type of dotted in the main plot (GR, Turbo, Spiral) and the second factor spraying with determination arginine subplot of concentrations. 300 mg/L and the third factor spraying with Bio-Catalyst Hortiboots10Sub subplot with concentrations (0,5, 10) ml/l and interaction between them in the specific qualities of the cherry Lycopersicon esculentum. L And evaluates the drip irrigation system carried out the working experiment according to the design of the full random sectors R.C.B.D and by arranging the system of dissident splinter panels Split Split Plot and four replication.The results show The amino acid arginine at a concentration of 300 mg/L was significantly superior to the content of lycopene and vitamin C in the fruits and the number of fruits amounted to 29.28 mg/100g, 17.22 mg/100gm, 76.84 fruits/plant compared to control, which amounted to 22.65 mg/100gm, 12.39mg/100gm, 65.18 fruits/plant As for the effect of spraying with humic acid, we notice that 10 ml/liter of humic acid has the highest percentage in the content of vitamin C in fruits, and the number of fruits reached 17.40 mg/100 g and 74.80 fruits/plant and for the effect of the emitter type, the Spiral was superior to the vitamin C content of fruits, which amounted to 21.00 mg/100 g, while the superiority of the emitter GR in the number of fruits was 85.97 fruits/plant.

Amino Acid Dependent Performance of Modified Chitosan Against Bacteria and Red Blood Cell.

In order to combat antibiotic resistance, the development of new antibacterial agents is essential. In this study, we prepared four types of amino acid modified chitosan (CS-AA). Compared with chitosan modified with hydrophobic amino acids, the chitosan modified with positively charged amino acids showed higher antibacterial efficiency against Escherichia coli (E. coli) under similar grafting rate. CS-AA achieves antibacterial properties mainly by destroying the integrity of bacterial cell membranes. All the four types of CS-AA show low toxicity towards red blood cells. This work indicates that positively charged groups are more important than hydrophobic groups in the design of chitosan-based antibacterial agents, and provides helpful information for the molecular design of effective antibacterial agents.

Removal performance and mechanism of typical amino acids in water by the peroxymonosulfate/Fe3O4 nanoparticles

Abstract Dissolved organic nitrogen (DON) as precursors of nitrogenous disinfection byproducts (N-DBPs) has become a serious issue for drinking water treatment. Here, Fe3O4/peroxymonosulfate (PMS) system was used to examine the amino acid removal and formation of N-DBPs in the system and the corresponding mechanisms. Results showed a remarked variation in removal efficiency of three typical amino acids, i.e., glutamate (78%), histidine (53%) and phenylalanine (27%) in Fe3O4/PMS system at optimum conditions (0.1 g/L Fe3O4, 1.5 mM PMS, 1 h). Notably, Fe3O4/PMS treatment led to dichloroacetonitrile (DCAN) formation caused by the chlorination of glutamate, phenylalanine and histidine being reduced by 53.3%, 9.7% and 41.9%, respectively. The degradation and subsequent N-DBPs formation in the Fe3O4/PMS system mainly depended on the types and properties of the amino acids. The formation of dichloroacetamide (DCAcAm) exhibited different trends, which may be due to the different R group structure of the three amino acids and the special aromaticity of the imidazole ring in the histidine side chain that facilitates its quick electrophilic substitution and ring-opening reaction. This study highlights that the Fe3O4/PMS system is a promising strategy to remove DON and efficiently eliminate N-DBPs formation in the drinking water treatment process depending on the amino acid type.

Morphometric, Histochemical, and Ultrastructural Analysis of the Reproductive System and Spermatogenic Stages of Male Blue Crab (Callinectessapidus Rathbun, 1896)

The blue crab, Callinectes sapidus Rathbun, 1896, is considered to be a luxury meal, especially in touristic cities. It contains more than 20 types of amino acids and provides all the needed amounts for human growth. This study describes the morphology and complex differentiation in the gonadosomatic index, morphological and ultrastructure features of the reproductive system, spermatogenesis, and spermatophores structure; this is due to the need to maintain natural and fishing stocks. Mature adult male crabs (carapace length 59 ± 7.12; width 126 ± 18.8 mm) were obtained from Abu-Qir Bay from November 2018 to October 2019 and transported alive in seawater to the laboratory. The reproductive system was dissected and weighed to the nearest 0.001g using the electronic balance, and the gonadosomatic index was subsequently calculated. The morphological analysis showed the developing testes with highly compacted seminiferous tubules. Using Periodic acid–Schiff stain, the spermatophore appeared with a zigzag-shaped wall that indicates its carbohydrate constituents. Each Spermatophore consisted of an inner spermatozoal mass embedded in a matrix, whose main components were secretions 1 and 2, and an outer thin acellular layer composed of secretions 3 and 4 from the anterior vas deferens (AVD). Secretions 5 and 6 (S5 and S6) also appeared with carbohydrate constituents using Mallory triple stain. The transverse section of the middle vas deferens (MVD) showed spermatophores with rod-shaped secretion S6 and granular secretions 7 (S7), forming a complex matrix between spermatophores. The secretion found in the MVD was granular, strongly acidophilic, and secreted by its highly columnar epithelium (S7). The ultrastructure showed that the testes were surrounded by a monolayer of myoid cells with an elongated nucleus, which also contained the following stages: spermatogonia, primary spermatocyte, secondary spermatocytes, and spermatids as well as spermatozoa. On the other hand, scanning electron microscope studies for fully formed spermatophore taken from the middle part of the vas deferens indicated that it is ellipsoidal in its outline with terminal stalk. Furthermore, the spermatophore was surrounded by a thick capsule of non-cellular substances and contained mature spermatozoa.

Nutritional composition and amino acid profile of gindara fish eggs (Lepidocybium sp.)

Gindara fish eggs are food that contains high nutrients, especially protein and other nutrients, and are used as local food for the coastal community. However, information about amino acids is not widely known. Therefore, this study aims to determine the amino acid profile of gindara fish eggs (Lepidocybium sp.). The method used is the exploratory method, and the test parameters include proximate analysis, amino acid profile, and protein efficiency ratio prediction. The water content of gindara fish eggs was 50.18%; protein 27.53%; fat 18.32%; ash 2.07% and 1.15% carbohydrates. Amino acids consist of 18 types of amino acids include 10 types of essential amino acids and 6 types of non-essential amino acids. The highest essential amino acid content of gindara fish eggs was 3.57% leucine, and the highest non-essential amino acid was glutamic acid at 4.79%. The major limiting amino acid in gindara fish eggs is tryptophan 0.25%, and the P-PER value of gindara fish eggs is 1.23%.

Exploring proteins in living cells

<p indent="0mm">Proteins have been studied for over <sc>200 years,</sc> but much of that work has been performed <italic>in vitro</italic>. This article briefly summarized the current status of protein exploration in living cells, and then presented some of my own representative findings in this field. First, I summarized the major methodologies hitherto developed for studying proteins <italic>in vivo</italic>, including visualizing a target protein by fusing it with the green fluorescent protein; investigating the tertiary structure of the target protein via in-cell nuclear magnetic resonance spectroscopy; and exploring the behaviors of a target protein by genetically labeling it with different types of unnatural amino acids. Next, I described how my laboratory applied and modified an <italic>in vivo</italic> methodology in which the genetic introduction of a photoactivatable, unnatural amino acid at a selected residue position in a target protein then mediates crosslinking of that protein with other proteins when the cells are irradiated by UV light. By improving that method, my laboratory contributed the following: (1) The creation of a bacterial strain with a genome that has a gene that encodes the orthogonal aminoacyl-tRNA synthetase and one that encodes the tRNA that are both needed to incorporate an unnatural amino acid (i.e., Bpa) into any target protein. (2) The development of a system in which the unnatural amino acid is randomly introduced at each of the target protein’s amino acid residue positions and, after detecting a meaningful interaction, DNA sequencing subsequently identifies that unnatural amino acid’s exact position. (3) The incorporation of the unnatural amino acid into a target protein by directly modifying the endogenous target gene in the genomic DNA so that the variant protein is produced spatiotemporally identical to that of the wild-type protein. (4) The development of a high-throughput polyacrylamide gel electrophoresis system in which 384 protein samples can be analyzed in a single gel. Finally, I described a few representative results from my lab by using <italic>in vivo</italic> protein photocrosslinking analysis: (1) The serendipitous discovery of a reversible subcellular structure (regrowth-delay body) that forms when a highly selected group of important proteins assembles only in non-dividing bacterial cells, and then dissolves when the cells resume growth. Their presence thus marks dormant persister bacterial cells that are known to tolerate antibiotics. (2) A revelation of a “gear shifting” mechanism that modulates bacterial ATP synthase activity via adaptation of its ε-subunit to the “inserted” or “non-inserted” conformation as the cell responds to a variety of favorable to unfavorable cellular conditions. (3) The identification of a supramolecular protein complex that is composed of proteins present in the cytoplasm, inner membrane, periplasm and outer membrane of Gram-negative bacteria, and that mediates the biogenesis of a group of outer membrane proteins. (4) The unveiling of a unique way that the acid stress chaperone HdeA acts. It exhibits biological activity while its tertiary structure becomes largely disordered, and then it protects other chaperones under acidic conditions. (5) A clarification that the long-designated bifunctional protein DegP acts primarily as a proteinase with little chaperone activity. (6) The discovery that small heat shock proteins are activated in a temperature-responsive, cascade manner that allows them to significantly increase their biological activity at high temperatures. I then conclude with a list of unresolved issues regarding protein exploration in living cells.

Enantiomeric purity deviations of radiolabelled amino acids obtained from chiral columns

Abstract Enantiomeric purity (EP) is an important value which denotes the relative percentage of the L-isomer with respect to the D-isomer. For 11C and 18F-labelled amino acid (AA) radiopharmaceutical (RP) production, EP represents a quality control parameter specified in European and national monographs for particular RPs. In most instances, EP value of greater then 90 or 95% (depending on AA type) is required as part of the quality control (QC) value of a RP following radiosynthesis. In common practice, two chromatographic columns are used for the EP determination of RPs: Crownpak CR(+) (Daicel), which contains a crown ether stationary phase or Chirobiotic T (Astec), which contains silica-bound glycoproteins as the stationary phase. The application of column Crownpak CR(+) requires that only perchloric acid solution (with pH 1–2) may be used, as the retention capability of the stationary phase is greatly reduced using organic solvents. This work intends to identify which chromatographic system is more accurate and reliable for EP determination as part of QC. We performed a series of parallel injections of the same batch of the widely used AA RPs [11C]MET and [18F]FET on the two aforementioned columns. The EP determination using column Crownpak CR(+) consistently provided a lower EP value compared to the Chirobiotic T column; the EP deviation between the respective columns was found to range from 2.4–4.0% for the same RP sample. Furthermore, the EP value was influenced by a sample’s dilution factor, e.g. the EP was observed to increase up to 1.5% when the radioactive sample had a fivefold dilution factor. This phenomenon was consistent for both Crownpak CR(+) and Chirobiotic T columns. Finally, a series of standard solutions of non-radioactive methionine with various ratios of L-and D-isomers was analyzed. The data obtained for non-radioactive methionine confirmed that column Crownpak CR(+) incorrectly provided a higher D-enantiomer concentration, whereas Chirobiotic T was found to provide a lower D-enantiomer concentration of the same sample. The deviation from the theoretical EP value was between 0.67 and 1.92%.

Seven Amino Acid Types Suffice to Create the Core Fold of RNA Polymerase.

The extant complex proteins must have evolved from ancient short and simple ancestors. The double-ψ β-barrel (DPBB) is one of the oldest protein folds and conserved in various fundamental enzymes, such as the core domain of RNA polymerase. Here, by reverse engineering a modern DPBB domain, we reconstructed its plausible evolutionary pathway started by "interlacing homodimerization" of a half-size peptide, followed by gene duplication and fusion. Furthermore, by simplifying the amino acid repertoire of the peptide, we successfully created the DPBB fold with only seven amino acid types (Ala, Asp, Glu, Gly, Lys, Arg, and Val), which can be coded by only GNN and ARR (R = A or G) codons in the modern translation system. Thus, the DPBB fold could have been materialized by the early translation system and genetic code.